Metal nanoparticles have a wide variety of applications in biomedical applications and in particular in biological labelling and detection techniques. For example, ligand-functionalised magnetic nanoparticles (for example iron oxide nanoparticles) are used in cell separation and probing (Ref: Pankhurst et al, J. Phys. D: Appl. Phys., 2003, 36, R167-R181) and gold nanoparticles have been widely used in immunohistochemistry to identify protein-protein interaction.
One technique which utilises metal nanoparticles is Surface Enhanced Raman Spectroscopy (SERS) and the related technique Surface Enhanced Resonance Raman Spectroscopy (SERRS). SERRS is an extraordinarily sensitive and information-rich spectroscopic method that relies upon the surface-enhancement of the in resonance Raman scattering of a reporter molecule (dye) localised on a suitable metallic substrate, e.g., silver or gold (Ref: Munro et al, Langmuir, 1995, 11, 3712). The present inventors have previously disclosed the use of SERRS techniques for the detection and identification of nucleic acid sequences. For example, WO 97/05280 and 99/60157 disclose methods and materials for detecting or identifying particular nucleic acid sequences in a sample using SERRS. The sample is exposed to a detection agent comprising a colloid metal surface associated with a SERRS active species (SAS) such as an azo dye, and with a target binding species (TBS) which is complementary to the target. The sample/agent mixture is observed to detect any surface enhancement of the label. Generally a surface-seeking group such as the benzotriazole group is used to promote chemisorption of the SAS and/or TBS to the metal surface.
In techniques using nanoparticles, it is often desirable to control the degree to which the metal nanoparticles are aggregated. In particular, in SERRS detection, the highest possible signal enhancements are realised only when the particles are aggregated, as it is the high electric field gradients generated at the junctions of the aggregated particles that are believed to be responsible for the highest signal enhancements (Ref: Moskovits, Rev. Mod. Phys., 1985, 57, 783). It is therefore advantageous to have a high degree of control over the extent to which aggregation occurs (Ref: Khan et al, Faraday Discuss., 2006, 132, 171).
Individual nanoparticles can be induced to aggregate by the addition of aggregating agents such as sodium chloride, or by the addition of dyes with an affinity for the metal surface. Previously, attempts have been made to encapsulate SERRS-active nanoparticle aggregates in polymer beads (Ref: McCabe et al, Faraday Discuss., 2006, 132, 303) to prevent further irreversible aggregation and thereby loss of signal intensity. However particle aggregation within each bead was variable. Besides this, no routine method for controlling the aggregation of metal nanoparticles, in particular SERRS-active metal nanoparticles, exists.
The control of aggregation of metal nanoparticles may also be of benefit in other analytical techniques. The benefits may include improvement in detection by plasmon resonance emission and absorption, and enhanced fluorescence. There are also a growing number of aggregation-dependent sensors that make use of a simple colour change in the presence of a target
In addition, increasing the stability of dye-loaded, aggregated metal nanoparticles is of interest in many fields, but in particular in SERRS techniques because a stable aggregation state gives rise to a stable SERRS response. This therefore significantly extends the range of potential applications.
In addition to the above, where it is desired to bind a chromophore to a metal surface (e.g. for use as a marker, or in an assay, and irrespective of aggregation) it will generally be desirable to increase surface adhesion of the chromophore so as to reduce leaching.
Metal surfaces other than nanoparticulate surfaces may also be used in SERRS and other techniques and protection and stabilisation of these surfaces is also of great interest. Another key problem is to avoid displacement of analytes from the surface of the metals on exposure to solutes, such as sodium chloride.